Hydantoin-containing glucokinase activators

ABSTRACT

Hydantoin compounds which are active as glucokinase activators to increase insulin secretion which makes them useful for treating type II diabetes.

[0001] This application claims priority under 35 U.S.C. § 119(e) ofprovisional application Serial No. 60/201,498, filed May 3, 2000.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is related toU.S. Ser. No. 09/526,143 filed Mar. 15, 2000 and U.S. Ser. No.09/532,506 filed Mar. 21, 2000. BACKGROUND OF THE INVENTION

[0002] Glucokinase (GK) is one of four hexokinases found in mammals[Colowick, S. P., in The Enzymes, Vol. 9 (P. Boyer, ed.) Academic Press,New York, N.Y., pages 1-48, 1973]. The hexokinases catalyze the firststep in the metabolism of glucose, i.e., the conversion of glucose toglucose-6-phosphate. Glucokinase has a limited cellular distribution,being found principally in pancreatic β-cells and liver parenchymalcells. In addition, GK is a rate-controlling enzyme for glucosemetabolism in these two cell types that are known to play critical rolesin whole-body glucose homeostasis [Chipkin, S. R., Kelly, K. L., andRuderman, N. B. in Joslin's Diabetes (C. R. Khan and G. C. Wier, eds.),Lea and Febiger, Philadelphia, Pa., pages 97-115, 1994]. Theconcentration of glucose at which GK demonstrates half-maximal activityis approximately 8 mM. The other three hexokinases are saturated withglucose at much lower concentrations (<1 mM). Therefore, the flux ofglucose through the GK pathway rises as the concentration of glucose inthe blood increases from fasting (5 mM) to postprandial (≈10-15 mM)levels following a carbohydrate-containing meal [Printz, R. G.,Magnuson, M. A., and Granner, D. K. in Ann. Rev. Nutrition Vol. 13 (R.E. Olson, D. M. Bier, and D. B. McCormick, eds.), Annual Review, Inc.,Palo Alto, Calif., pages 463-496, 1993]. These findings contributed overa decade ago to the hypothesis that GK functions as a glucose sensor inβ-cells and hepatocytes (Meglasson, M. D. and Matschinsky, F. M. Amer.J. Physiol. 246, E1-E13, 1984). In recent years, studies in transgenicanimals have confirmed that GK does indeed play a critical role inwhole-body glucose homeostasis. Animals that do not express GK diewithin days of birth with severe diabetes while animals overexpressingGK have improved glucose tolerance (Grupe, A., Hultgren, B., Ryan, A. etal., Cell 83, 69-78, 1995; Ferrie, T., Riu, E., Bosch, F. et al., FASEBJ., 10, 1213-1218, 1996). An increase in glucose exposure is coupledthrough GK in β-cells to increased insulin secretion and in hepatocytesto increased glycogen deposition and perhaps decreased glucoseproduction.

[0003] The finding that type II maturity-onset diabetes of the young(MODY-2) is caused by loss of function mutations in the GK gene suggeststhat GK also functions as a glucose sensor in humans (Liang, Y.,Kesavan, P., Wang, L. et al., Biochem. J. 309, 167-173, 1995).Additional evidence supporting an important role for GK in theregulation of glucose metabolism in humans was provided by theidentification of patients that express a mutant form of GK withincreased enzymatic activity. These patients exhibit a fastinghypoglycemia associated with an inappropriately elevated level of plasmainsulin (Glaser, B., Kesavan, P., Heyman, M. et al., New England J. Med.338, 226-230, 1998). While mutations of the GK gene are not found in themajority of patients with type II diabetes, compounds that activate GKand, thereby, increase the sensitivity of the GK sensor system willstill be useful in the treatment of the hyperglycemia characteristic ofall type II diabetes. Glucokinase activators will increase the flux ofglucose metabolism in β-cells and hepatocytes, which will be coupled toincreased insulin secretion. Such agents would be useful for treatingtype II diabetes.

SUMMARY OF THE INVENTION

[0004] This invention provides a compound, comprising a substitutedhydantoin of the formula:

[0005] wherein

[0006] R₁ is a five- or six- membered aromatic heterocyclic ring havingone to three heteroatoms selected from nitrogen, oxygen, and sulfur,which ring is unsubstituted or substituted with halo, amino,hydroxylamino, nitro, cyano, sulfonamido, lower alkyl, perfluoro loweralkyl, lower alkyl thio, perfluoro-lower alkyl thio, lower alkylsulfonyl, perfluoro-lower alkyl sulfonyl, lower alkyl sulfinyl, or—(R₅)_(n)-C(O)—OR₆;

[0007] R₂ is a cycloalkyl ring containing from 5 to 7 carbon atoms;

[0008] R₃ is hydrogen, lower alkyl, a cycloalkyl ring containing from 5to 7 carbon atoms, unsubstituted aryl, aryl substituted with halo orhydroxy, or an unsubstituted five- or six- membered aromaticheterocyclic ring having one or two heteroatoms selected from nitrogen,oxygen, and sulfur;

[0009] R₄ is hydrogen, lower alkyl, or R₃ and R₄ together with thecarbon atom to which they are attached form a cycloalkyl ring containing5 to 7 carbon atoms;

[0010] R₅ is —C(O)— or lower alkyl;

[0011] R₆ is lower alkyl;

[0012] n is 0 or 1; * and ** each designate an asymmetric centers, andpharmaceutically acceptable salts thereof.

[0013] The compounds of Formula I have been found to activateglucokinase. Glucokinase activators are useful for increasing insulinsecretion in the treatment of type II diabetes. Therefore compounds ofthis invention are useful to increase insulin secretion in view of theiractivity as glucokinase activators.

DETAILED DESCRIPTION OF THE INVENTION

[0014] This invention is directed to compounds of Formula I above. Theinvention is particularly directed to compounds as follows, where:

[0015] R₂ and R₃ are both cyclohexyl, or

[0016] R₃, when it is lower alkyl, is methyl, ethyl, propyl, or butyl,or

[0017] R₄, when it is lower alkyl, is methyl or ethyl (especiallycompounds where R₃ and R₄ are both so defined), or

[0018] R₁, when substituted, is substituted with halo, lower alkyl, or—(R₅)_(n)-C(O)—OR₆, or compounds where any two or more, or all, of theseconditions are met. For any compound of this invention where R₁, R₂, orR₃ are not specified, it is preferred that the variable is as describedin this paragraph.

[0019] Certain preferred compounds of Formula I include a compound whereR₁ is substituted or unsubstituted thiazolyl (Compound A). Among theembodiments of Compound A are those compounds where R₁ is thiazolylsubstituted with halo, lower alkyl, or —(R₅)_(n)-C(O)—OR₆, andespecially with —(R₅)_(n)-C(O)—OR₆. (Compound A-1). In Compound A-1, itis preferred that R₂ is cyclopentyl or cyclohexyl. It is also preferredthat R₃ is cyclopentyl or cyclohexyl. It is preferred that R₄ ishydrogen. It is especially preferred that R₂ and R₃ are cyclohexyl.

[0020] In preferred embodiments of Compound A-1, R₂ and R₃ arecyclopentyl or cyclohexyl, and R₄ is hydrogen (Compound A-1a). In oneembodiment of Compound A-1 a, n is 0 (e.g., the thiazolyl is substitutedwith —C(O)—OR₆). Examples of such compounds are

[0021](S,S)-2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0022](S,S)-2-[[2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl)-3-cyclopentylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0023](S,S)-2-[[3-cyclopentyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0024](S,S)-2-[[3-cyclohexyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0025] In such a compound, R₂ and R₃ may both be cyclohexyl, for example(S,S)-2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.

[0026] In another embodiment of Compound A-1a, R₅ is —C(O)— or loweralkyl (e.g., the thiazolyl is substituted with —C(O)—C(O)—OR₆ or -loweralkyl-C(O)—OR₆). In addition, in such compounds R₂ and R₃ may becyclohexyl. Examples of such compounds are

[0027](S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-yl]oxoaceticacid ethyl ester and

[0028](S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-yl]aceticacid ethyl ester.

[0029] In another embodiment of Compound A1, R₂ is cyclopentyl orcyclohexyl (Compound A-1b). In one embodiment of Compound A-1b, R₃ issubstituted or unsubstituted phenyl and R₄ is hydrogen. Examples ofthese compounds are

[0030](S,S)-2-[[2-(4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0031]2-[[(S)-2-[(R)-4-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-yl]-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0032](S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(4-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0033](S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(3-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester, and

[0034]2-[[(S)-3-cyclohexyl-2-[(R,S)-2,5-dioxo-4-(4-fluorobenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0035] In another embodiment of Compound A-1b, at least one of R₃ and R₄are lower alkyl. Examples of such compounds are

[0036](S)-2-[[3-cyclohexyl-2-(4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester, and

[0037]2-[[(S)-3-cyclohexyl-2-[(R)-2,5-dioxo-4-propylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0038] In yet another embodiment of Compound A-1b, R₃ is naphthyl and R₄is hydrogen. An example of such a compound is(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(naphthalen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0039] In another embodiment of Compound A-1b, R₃ and R₄ together withthe carbon atoms to which they are attached form a cycloalkyl ringcontaining 5 to 7 carbon atoms. An example of such a compound is(S)-2-[[3-cyclohexyl-2-(2,4-dioxo-1,3-diazaspiro[4.4]non-3-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0040] And in another embodiment of Compound A-1b, R₃ is anunsubstituted five- or six-membered aromatic heterocyclic ring havingone or two heteroatoms selected from nitrogen, oxygen, and sulfur. Anexample of such a compound is(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(thiophen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0041] In one embodiment of Compound A (a compound of Formula I whereinR₁ is substituted or unsubstituted thiazolyl), R₁ is unsubstitutedthiazolyl (Compound A-2). It is preferred that R₂ and R₃ are cyclohexyland R₄ is hydrogen. An example of such a Compound A-2 is(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(thiazole-2-yl)propanamide.

[0042] In other preferred compounds of Formula I, R₁ is substituted orunsubstituted pyridine (Compound B). It is preferred that R₂ iscyclopentyl or cyclohexyl, especially cyclohexyl. It is also preferredthat R₃ is cyclopentyl or cyclohexyl, especially cyclohexyl. It ispreferred that R₄ is hydrogen.

[0043] In one embodiment of Compound B, R₂ is cyclohexyl. In such acompound where R₂ is cyclohexyl, it is preferred that R₃ is cyclohexyland R₄ is hydrogen (Compound B-1).

[0044] In one embodiment of Compound B-1, R₁ is substituted pyridine.Preferably the pyridine is substituted with —(R₅)_(n)-C(O)—OR₆,especially where n is 0 and R₆ is lower alkyl, such as methyl (e.g.,methoxycarbonyl). Examples of such compounds are

[0045](S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(5-methylpyridin-2-yl)propanamide.

[0046](S,S)-6-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]nicotinicacid methyl ester, and

[0047](S,S)-N-(5-chloropyridin-2-yl)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanamide.

[0048] In another embodiment of Compound B-1, R₁ is unsubstitutedpyridine. An example of such a compound is (S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(pyridin-2-yl)propanamide.

[0049] In the compound of Formula I the “*” and “**” illustrate the twoseparate asymmetric centers. The (S) enantiomer at the positiondesignated by “**” is preferred. However the compounds of this inventionmay be pure (R)(R), pure (S)(S), pure (R)(S), pure (S)(R) or any mixtureof pure enantiomers.

[0050] As used throughout this application unless otherwise specified,the term “lower alkyl” includes both straight chain and branched chainalkyl groups having from 1 to 6 or 1 to 7 carbon atoms, such as methyl,ethyl, propyl, isopropyl, preferably methyl and ethyl. Unless otherwisespecified, propyl is taken to include both forms of propyl (e.g.,isopropyl, n-propyl) and butyl is taken to include all forms of butyl(e.g., isobutyl, n-butyl, tert-butyl). Preferred at R₃ is methyl, ethyl,propyl, or butyl. Preferred at R₄ is methyl or ethyl.

[0051] The term “cycloalkyl ring” may be a ring of from three to sevencarbon atoms, but preferably from five to seven carbon atoms, especiallycyclopentyl, cyclohexyl, cyclobutyl and cyclopropyl. The more preferablecycloalkyl groups contain from 5 to 6 carbon atoms, e.g., cyclopentyland cyclohexyl, and cyclohexyl is most preferable. As used herein,“perfluoro-lower alkyl” means any lower alkyl group wherein all of thehydrogens of the lower alkyl group are substituted or replaced byfluoro. Among the preferred perfluoro-lower alkyl groups aretrifluoromethyl, pentafluoroethyl, heptafluoropropyl, etc.

[0052] As used herein, “lower alkyl thio” means a lower alkyl group asdefined above where a thio group is bound to the rest of the molecule.Similarly “perfluoro-lower alkyl” thio means a perfluoro-lower alkylgroup as defined above where a thio group is bound to the rest of themolecule. As used herein, “lower alkyl sulfonyl” or “lower alkylsulfinyl” means a lower alkyl group as defined above where a sulfonyl orsulfinyl group is bound to the rest of the molecule. Similarly“perfluoro-lower alkyl sulfonyl” means a perfluoro-lower alkyl group asdefined above where a sulfonyl group is bound to the rest of themolecule.

[0053] When R₃ and R₄ together with the carbon atom to which they areattached form a cycloalkyl ring containing five to seven carbon atoms,this includes the ring carbon atom and the methylene linking the ringcarbon atom and R₄ such that if R₃ and R₄ are each methylene, cyclobutylis formed. If R₃ is methylene and R₄ is ethylene, cyclopentyl is formed,etc.

[0054] As used herein, the terms “halogen” or “halo” unless otherwisespecified, designates all four halogens, i.e. fluorine, chlorine,bromine and iodine.

[0055] R₁ is, and R₃ can be any five- or six-membered aromaticheterocyclic ring containing from one to three, preferably from one totwo, heteroatoms selected from the group consisting of sulfur, oxygen ornitrogen. Any such five- or six-membered aromatic heterocyclic ring canbe used in accordance with this invention. Among the preferred rings forR₁ are thiazole and pyridine (especially pyridine), and a preferred ringfor R₃ is thiophene. R₁, and R₃ when R₃ is a heterocyclic ring, isconnected to the remainder of the molecule of Formula I through a ringcarbon atom. When R₁ is substituted as described in Formula I, thesubstituent is on a ring carbon atom. R₁ is preferably monosubstituted,but may be di or tri substituted. A preferred substituent, especiallyfor pyridine, is lower alkoxy (preferably methoxy) carbonyl.

[0056] As used herein the term “aryl” signifies an aromatic hydrocarbonring having six or ten carbon atoms such as phenyl or naphthyl.

[0057] The compounds of this invention may be produced by the reactionschemes provided below.

[0058] The term “resin” designates any conventional polymer resin whichhas suitable characteristics for use in solid phase peptide synthesis. Aresin with the suitable characteristics is inert, physically stable,insoluble in inorganic solvents, and has a linker functionality which islabile under known chemical conditions. Preferred are polystyrene resinshaving chemically labile functional linkers such as trityl resins andespecially Wang resins.

[0059] The term “amino protecting group” designates any conventionalamino protecting group which can be cleaved to yield the free aminogroup. The preferred protecting groups are the conventional aminoprotecting groups utilized in peptide synthesis. Especially preferredare those amino protecting groups which are cleavable under treatmentwith secondary dialkyl amines. A particularly preferred amino protectinggroup is 9H-fluoren-9-ylmethoxy carbamate.

[0060] “Orthogonal” is the term used to describe the relationship of theamino protecting group to the resin. The resin and the amino protectinggroup must be compatible, in that the resin-peptide bond and the aminoprotecting group should not labile under the same conditions. Duringsynthesis of a given compound, one should be able to cleave the aminoprotecting groups off the compound while leaving the compound attachedto the resin. In other words, the conditions under which the aminoprotecting group comes off the compound should not also cause thecompound to come off the resin. It is preferred that the aminoprotecting group be cleavable under basic or weakly acidic conditions,because the preferred Wang-type resins are cleavable under stronglyacidic conditions (i.e. about pH 0 to about pH 1) A skilled person willreadily be able to determine the necessary conditions to select anorthogonal amino protecting group-resin set.

[0061] The term “pharmaceutically acceptable salts” as used hereininclude any salt with both inorganic or organic pharmaceuticallyacceptable acids such as hydrochloric acid, hydrobromic acid, nitricacid, sulfuric acid, phosphoric acid, citric acid, formic acid, maleicacid, acetic acid, succinic acid, tartaric acid, methanesulfonic acid,para-toluene sulfonic acid and the like. The term “pharmaceuticallyacceptable salts” also includes any pharmaceutically acceptable basesalt such as amine salts, trialkyl amine salts and the like. Such saltscan be formed quite readily by those skilled in the art using standardtechniques.

[0062] In accordance with this invention, the compounds of Formula I areproduced by the following reaction schemes. Any compound of Formula Imay be produced as shown in Reaction Scheme 1. The compounds of FormulaI-A are produced as shown in Reaction Scheme 2. Reaction Scheme 3 showshow to produce N-Fmoc-aminothiazole-4-carboxylic acid, which is compound3 of Scheme 2 where PG is the protecting group Fmoc.

[0063] wherein R₁, R₂, R₃ and R₄ are as previously described and PG₁ andPG₂ are amine protecting groups which may or may not be equivalent, thatare removable under conditions compatible with the Linker-O bond.

[0064] wherein R₂, R₃, R₄ and R₆ are as previously described and PG, PG₁and PG₂ are amine protecting groups which may or may not be equivalent,that are removable under conditions compatible with the linker-O bondand where the ring A represents a five or six membered heteroaromaticring having one, two or three hetero atoms selected from nitrogen,oxygen or sulfur.

[0065] The synthesis of the compounds of this invention may be carriedout by a procedure whereby each amino acid in the desired sequence isadded one at a time in succession to another amino acid or residuethereof or by a procedure whereby peptide fragments with the desiredamino acid sequence are first synthesized conventionally and thencondensed to provide the compound.

[0066] Such conventional procedures for synthesizing the novel compoundsof the present invention include for example any solid phase peptidesynthesis method. In such a method the synthesis of the novel compoundscan be carried out by sequentially incorporating the desired amino acidresidues one at a time into the growing peptide chain according to thegeneral principles of solid phase methods [Merrifield, R. B., J Amer.Chem. Soc. 1963, 85, 2149-2154; Barany et al., The Peptides, Analysis,Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds.Academic Press 1-284 (1980); Bunin, B., Combinatorial Index, AcademicPress (1998)].

[0067] Common to chemical syntheses of peptides is the protection ofreactive side chain groups of the various amino acid moieties withsuitable protecting groups, which will prevent a chemical reaction fromoccurring at that site until the protecting group is ultimately removed.Usually also common is the protection of the alpha amino group of anamino acid or fragment while that entity reacts at the carboxyl group,followed by the selective removal of the alpha amino protecting groupand allow a subsequent reaction to take place at that site. Whilespecific protecting groups are mentioned below in regard to the solidphase synthesis method, it should be noted that each amino acid can beprotected by any protective group conventionally used for the respectiveamino acid in solution phase synthesis.

[0068] For example, alpha amino groups may be protected by a suitableprotecting group selected from aromatic urethane-type protecting groups,such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz);aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, andallyloxycarbonyl. In the present case, Fmoc is the most preferred foralpha amino protection. Guanidino groups may be protected by a suitableprotecting group selected from nitro, p-toluenesulfonyl (Tos), Z,pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, and Boc. Pmc isthe most preferred for arginine (Arg).

[0069] The solvents dichloromethane, dimethylformamide (DMF) andN-methylpyrrolidinone and toluene may be purchased from Fisher orBurdick and Jackson and may be used without additional distillation.Trifluoroacetic acid was purchased from Halocarbon or Fluka and usedwithout further purification. Diisopropylcarbodiimide anddiisopropylethylamine (DIPEA) was purchased from Fluka or Aldrich andused without further purification. 1 -Hydroxybenzotriazole (HOBT) may bepurchased from Sigma Chemical Co. and used without further purification.Protected amino acids, unless otherwise specified, are generallypreferably of the L configuration and may be obtained commercially fromBachem, Advanced ChemTech, or Neosystem. Such amino acids may also bechemically synthesized using any one of several well known methods ofamino acid synthesis. The configuration of the amino acids 5 and 7 usedto prepare a given compound of this invention will determine theconfiguration of the ** and * positions respectively of Formula I.Therefore, it is useful to select the amino acid configuration with thedesired final configuration in mind. L amino acids have the (S) absoluteconfiguration and D amino acids have the (R) absolute configuration.

[0070] Compounds of this invention may be prepared using solid phasesynthesis following the principles and general methods described byMerrifield or by Bunin, although other equivalent chemical synthesisknown in the art could be used as previously mentioned. Solid phasesynthesis is commenced from the C-terminal end of the peptide bycoupling a N-protected amino acid to a suitable resin. Such a startingmaterial can be prepared by attaching an N-protected amino acid by anester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, or by anamide bond between an Fmoc-Linker, such asp-[(R,S)-α-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyaceticacid (Rink linker) to a benzhydrylamine (BHA) resin. Preparation of thehydroxymethyl resin is well known in the art. Wang resin supports arecommercially available and generally used when the desired peptide beingsynthesized has an ester or a substituted amide at the C-terminus. Toform the starting resin bound amino acid, a Fmoc N-protected amino acidis activated by the formation of a mixed anhydride which in turn coupleswith the hydroxymethyl resin though an ester bond. Several reagents areused to form mixed anhydrides in which the carbonyl group originatingfrom the C-terminal amino acid is preferentially activated tonucleophilic attack by the hydroxymethyl residues in the Wang resin,through either electronic or steric effects. For example, appropriatecompounds used in the formation of the mixed anhydrides aretrimethylacetyl chloride, 2,6-dichlorobenzoyl chloride and2,4,6-trichlorobenzoyl chloride, preferably 2,6-dichlorobenzoylchloride.

[0071] Subsequently, the amino acids or mimetics are then coupled ontothe Wang resin using the Fmoc protected form of the amino acid ormimetic, with 2-5 equivalents of amino acid and a suitable couplingreagent. After each coupling, the resin may be washed and dried undervacuum. Loading of the amino acid onto the resin may be determined byamino acid analysis of an aliquot of Fmoc-amino acid resin or bydetermination of Fmoc groups by UV analysis.

[0072] The resins are carried through one or two cycles to add aminoacids sequentially. In each cycle, the N-terminal Fmoc protecting groupis removed under basic conditions from the resin bound amino acid. Asecondary amine base such as piperidine, piperazine or morpholine,preferably piperidine (20-40% v/v) in an inert solvent, for example,N,N-dimethylformamide is particularly useful for this purpose. Followingthe removal of the alpha amino protecting group, the subsequentprotected amino acids are coupled stepwise in the desired order toobtain an N-Fmoc protected peptide-resin. The activating reagents usedfor coupling of the amino acids in the solid phase synthesis of thepeptides are well known in the art. For example, appropriate couplingreagents for such syntheses are[(benzotriazol-1-yl)oxy]tris(dimethylamino) phosphoniumhexafluorophosphate (BOP), [(benzotriazol-1-yl)oxy]tis(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP),O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), and diisopropylcarbodiimide (DIC),preferably HBTU and DIC. Other activating agents as described by Baranyand Merrifield [The Peptides, Vol. 2, J. Meienhofer, ed., AcademicPress, 1979, pp 1-284] may be utilized. The couplings are convenientlycarried out in an inert solvent, such as N,N-dimethylformamide orN-methylpyrrolidinone, preferably N-methylpyrrolidinone, optionally inthe presence of a substance that minimizes racemization and increasesthe rate of reaction. Among such substances are 1-hydroxybenzotriazole(HOBT), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT),1-hydroxy-7-azabenzotriazole (HOAT), and N-hydroxysuccinimide (HOSu). Inthe present instance, HOBT is preferred.

[0073] The protocol for a typical coupling cycle is as follows (MethodB): Step Reagent Time 1 20% piperidine/DMF 30 mm 2 DMF 3 × 30 sec 3methanol 3 × 30 sec 4 dichloromethane 3 × 30 sec 5 coupling overnight 6DMF 3 × 30 sec 7 methanol 3 × 30 sec 8 dichloromethane 3 × 30 sec

[0074] Solvents for all washings and couplings may be measured tovolumes of, for example, 10-20 ml/g resins. Coupling reactionsthroughout the synthesis may be monitored by assays, such as the Kaiserninhydrin test, to determine extent of completion [Kaiser et at. Anal.Biochem. 1970, 34, 595-598].

[0075] When the requisite number of amino acid units have been assembledon the resin, the N-terminal Fmoc group may be cleaved using Steps 1-4of Method B and the deprotected amine is reacted with phosgene or aphosgene equivalent to form an isocyanate. The reagent of choice in thistransformation is trichloromethyl chloroformate (diphosgene). Thereaction is carried out in an inert solvent, for exampledichloromethane, in the presence of a proton acceptor. When a suspensionof the resin bound isocyanate is heated, cyclization occurs wherein theisocyanate moiety condenses with the nitrogen of the neighboring amidegroup to form a 2,5-dioxoimidazolidine ring.

[0076] The compounds may be cleaved from the resin by the followingprocedure, conditions which also remove other protecting groups if theyare present. The peptide-resins are shaken in a mixture (1:1) oftrifluoroacetic acid in dichloromethane, optionally in the presence of acation scavanger, for example ethanedithiol, dimethylsulfide, anisole ortriethylsilane, at room temperature for 60 min. The cleavage solutionmay be filtered free from the resin, concentrated to dryness, and theproduct then used per se in subsequent transformations as shown inReaction Scheme 1 and Reaction Scheme 2.

[0077] Compounds of Formula 1 can be prepared by the methods outlined inReaction Scheme 1 and Reaction Scheme 2. Reaction Scheme 2 is a generalprocedure that can be used to prepare all compounds embodied by Formula1, but in the present case, it is particularly useful in the preparationof compounds where R₁ is varied while R₂ and R₃ are limited tocycloalkyl and R₄ is hydrogen. Reaction Scheme 1 is used in thepreparation of compounds of Formula I-A.

[0078] In Reaction Scheme 2, an N-protected-amino acid 3 (see ReactionScheme 3) is converted to a mixed anhydride on treatment with2,6-dichlorobenzoyl chloride in the presence of Wang resin 2 and aproton acceptor, such as triethylamine, diisopropylethylamine orpyridine, preferably pyridine to give the resin bound amino acid ofstructure 4. The reaction is conveniently carried out in an inertsolvent for example N,N-dimethylformamide or N-methylpyrrolidinone,preferably N-methylpyrrolidinone at from zero degrees to roomtemperature, most conveniently at room temperature. The conversion of 4to the resin bound compound of structure 6 can be achieved by using theprotocol outlined in method B. Thus after N-deprotection of the resinbound amino acid of structure 4 with piperidine inN,N-dimethylformamide, the product is then acylated with theN^(α)-protected amino acid of structure 5 in the presence ofdiisopropylcarbodiimide and HOBT in N-methylpyrrolidinone. Thedeprotection and N-acylation is carried out at a temperature betweenabout zero degrees and about room temperature, preferably at about roomtemperature. By using the coupling cycle described above for theconversion of 4 to 6 the N^(α)-protected amino acids of structure 7 isincorporated into the resin bound compound of structure 6. Thuscompounds of structure 6 are sequentially deprotected with piperidine inN,N-dimethylformamide and then coupled with compounds of structure 7 inthe presence of diisopropylcarbodiimide and 1-hydroxybenzotriazole inN-methylpyrrolidinone at a temperature between about zero degrees andabout room temperature, preferably at about room temperature to affordthe resin bound compounds of structure 8.

[0079] The N-terminus protecting group PG₂ in the compounds of structure8 was removed on treatment with a secondary amine base, preferablypiperidine in an inert solvent (preferably N,N-dimethylformamide) andthen was reacted with phosgene or a phosgene equivalent reagent, toultimately yield in a two step sequence, the 2,5-dioxoimidazolidines ofstructure 10. The reaction to give the intermediate isocyanate 9 isconveniently carried out using trichloromethyl chloroformate(diphosgene) in an inert solvent, for example, a halogenated hydrocarbonin the presence of a proton acceptor, for example, pyridine,triethylamine or diisopropylethylamine, preferably diisopropylethylamineat a temperature between about zero degrees and about room temperature,preferably at about room temperature. The thermally induced cyclizationof the intermediate isocyanates is performed by heating a suspension ofthe resin bound isocyanates of structure 9 in an inert solvent, forexample toluene, at a temperature of from between 50° C. and the refluxtemperature of the mixture, preferably at about 70° C. to give the resinbound compounds of structure 10.

[0080] Cleavage of the assembled peptidic residue 10 from the solidsupport to give the acids of structure 11 is achieved by shaking asuspension of 10 in a strong acid, for example methanesulfonic acid,hydrofluoric acid or trifluoroacetic acid, preferably trifluoroaceticacid optionally in the presence of a cation scavenger and an inertco-solvent, for example dichloromethane. The reaction is convenientlyrun at a temperature between about zero degrees and about roomtemperature, preferably at about room temperature.

[0081] To complete the synthesis, the acid of structure 11 is reactedwith an alcohol (R₆OH) to form the ester 1. The esterification can beaccomplished using many of the methods well known to those of averageskill in the field of organic chemistry. The conversion is convenientlycarried out using a coupling reagent, for example one of the many usefulcarbodiimides, preferably the water soluble1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, optionally using R₆OH ora mixture of R₆OH and a inert co-solvent, e.g., dichloromethane, as thereaction medium. The reaction is run at a temperature between about zerodegrees and about room temperature, preferably at about roomtemperature.

[0082] In a similar fashion, Reaction Scheme 1, the N^(α)-protectedamino acids of structure 5 is converted to a mixed anhydride ontreatment with 2,6-dichlorobenzoyl chloride in the presence of Wangresin 2 and a proton acceptor, such as triethylamine,diisopropylethylamine or pyridine, preferably pyridine to give the resinbound amino acid of structure 12. The reaction is conveniently carriedout in an inert solvent for example N,N-dimethylformamide orN-methylpyrrolidinone, preferably N-methylpyrrolidinone at from zerodegrees to room temperature, most conveniently at room temperature. Theconversion of 12 to the resin bound compound of structure 13 can beachieved by using the protocol outlined in method B. Thus afterN-deprotection of the resin bound amino acid of structure 12 withpiperidine in N,N-dimethylformamide, the product is then acylated withthe N-protected amino acid of structure 7 in the presence ofdiisopropylcarbodiimide and HOBT in N-methylpyrrolidinone. Thedeprotection and N-acylation is carried out at a temperature betweenabout zero degrees and about room temperature, preferably at about roomtemperature.

[0083] The N-terminus protecting group PG₂ in the compounds of structure13 was removed on treatment with a secondary amine base, preferablypiperidine in an inert solvent, preferably N,N-dimethylformamide andthen was reacted with phosgene or a phosgene equivalent reagent, toultimately yield in a two step sequence, the 2,5-dioxoimidazolidines ofstructure 15. The reaction to give the intermediate isocyanate 14 isconveniently carried out using trichloromethyl chloroformate(diphosgene) in an inert solvent, for example, a halogenated hydrocarbonin the presence of a proton acceptor, for example, pyridine,triethylamine or diisopropylethylamine, preferably diisopropylethylamineat a temperature between about zero degrees and about room temperature,preferably at about room temperature. The thermally induced cyclizationof the intermediate isocyanates is accomplished by heating a suspensionof the resin bound isocyanates of structure 14 in an inert solvent, forexample toluene, at a temperature of from between 50° C. and the refluxtemperature of the mixture, preferably at about 70° C. to give the resinbound compounds of structure 15.

[0084] Cleavage of the peptidic residue 15 from the solid support togive the acids of structure 16 is achieved by shaking a suspension of 15in a strong acid, for example methanesulfonic acid, hydrofluoric acid ortrifluoroacetic acid, preferably trifluoroacetic acid optionally in thepresence of a cation scavenger and an inert co-solvent, for exampledichloromethane. The reaction is conveniently run at a temperature ofbetween about zero degrees and about room temperature, preferably atabout room temperature.

[0085] Reaction of the acid 16 with R₁-NH₂ to form the amide of Formula1 can be carried out under the coupling conditions previously described.The preferred coupling reagent in this instance is HBTU. The reaction iscarried out in the presence of a tertiary amine base, such astriethylamine or diisopropylethylamine, preferably diisopropylethylaminein an inert solvent, for example N,N-dimethylformamide orN-methylpyrrolidinone, preferably N-methylpyrrolidinone at from zerodegrees to room temperature, most conveniently at room temperature.

[0086] Reaction Scheme 3 outlines the preparation of the intermediateN-Fmoc-2-aminothiazole-4-carboxylic acid 3. Initially9-fluorenylmethoxycarbonyl chloride (18 ) is reacted with potassiumthiocyanate in an inert solvent, preferably ethyl acetate at atemperature of between zero degrees and 5° C. Then the reaction isallowed to proceed at a temperature of from zero degrees to 40° C.,preferably at room temperature to furnish N-Fmoc-thiocyanate (19 ).Treatment of 19 with a solution of ammonia in an inert solvent, forexample methanol or ethanol, preferably methanol at a temperature offrom zero degrees to room temperature, preferably zero degrees affordedN-Fmoc-thiourea 20. In the final step, the thiourea 20 is then reactedwith bromopyruvic acid to form the thiazole of structure 3. The reactionis conveniently carried out if an inert solvent, such as a cyclic ether,for example tetrahydrofuran or dioxane, preferably dioxane at atemperature of from 40° C. to the reflux temperature of the mixturepreferably at about 70° C.

[0087] All of the compounds of Formula I which include the compounds setforth in the Examples, activated glucokinase in vitro by the procedureof Example A. In this manner, they increase the flux of glucosemetabolism which causes increased insulin secretion. Therefore, thecompounds of Formula I are glucokinase activators useful for increasinginsulin secretion.

SYNTHESIS EXAMPLES

[0088] These examples are provided in illustration and are not intendedto limit the invention in any way.

[0089] Analytical high performance liquid chromatography was (HPLC) wasconducted on a Hewlett-Packard 1090 system with ultraviolet (UV)detection system at 214 nm using an ES Industries C₁₈ column (30×3.2mm). Preparative HPLC separations were carried out using a Shimazu VPseries system interfaced with a Perkin-Elmer Sciex Mass Spectrometerdetector (PE Sciex 150EX) using a YMC C₁₈ column (2×5 cm).

EXAMPLE 1

[0090] Preparation of N-Fmoc-thiourea.

[0091] To a suspension of potassium thiocyanate (8.55 g, 88 mmol) inethyl acetate (100 mL) cooled to 0° C. was added dropwise a solution of9-fluorenylmethoxycarbonyl chloride (20.7 g, 80 mmol) in ethyl acetate(100 mL) over a period of 15 min. The resulting suspension was allowedto warm to ambient temperature overnight with stirring. The formed solidwas filtered off and the filtrate was concentrated in vacuo to afford anorange oil. Without further purification, the oil was dissolved inethanol (50 mL) and treated by dropwise addition with a cold solution ofammonia in ethanol (7N, 91 mL, 637 mmol). A precipitate formed uponaddition of the ammonia solution. The suspension was stirred vigorouslyat 0° C. for 15 min and then the solids were filtered off, washed withcold ethanol (3×20 mL) and dried to afford N-Fmoc-thiourea (16.8 g, 70%)as an off-white solid: EI-HRMS m/e calcd for C₁₆H₁₄N₂O₂S (M⁺) 298.0776,found 298.0770.

EXAMPLE 2

[0092] Preparation of N-Fmoc-2-aminothiazole-4-carboxylic acid.

[0093] A solution of N-Fmoc-thiourea (5.96 g, 20 mmol) in dioxane (40mL) was treated with bromopyruvic acid (3.34 g, 20 mmol). The reactionmixture was refluxed for 1 h, then the precipitated solids wererecovered by filtration and washed with diethyl ether (3×20 mL) toafford N-Fmoc-2-aminothiazole-4-carboxylic acid (7.1 g, 97%) as a whitesolid: EI-HRMS m/e calcd for C₁₉H₁₄N₂O₄S (M⁺) 366.0674, found 366.0679.

EXAMPLE 3

[0094] Preparation of (S)-2-[[3-cyclohexyl-2-(4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0095] Step (i). A mixture of N-Fmoc-2-aminothiazole-4-carboxylic acid(6.0 g, 16.5 mmol), 2,6-dichlorobenzoyl chloride (7.9 mL, 55 mmol) inN-methylpyrrolidinone (50 mL) was added into a fritted polypropylenecolumn charged with Wang resin (Midwest Bio-Tech, 10 g, 11 mmol). Afterthe suspension was shaken for 5 min, pyridine (6.2 mL, 77 mmol) wasadded slowly and the resulting dark mixture was shaken overnight atambient temperature. The mixture was then filtered and the resin waswashed with N,N-dimethylformamide (3×100 mL), methanol (3×100 mL),dichloromethane (3×100 mL) and dried in vacuo.

[0096] Step (ii). To the resin product of the previous step (3 g, 2.31mmol) was added 20% piperidine in N,N-dimethylformamide (25 mL). Thereaction mixture was shaken at ambient temperature for 30 min. Themixture was filtered and the resin was washed with N,N-dimethylformamide(3×30 mL), methanol (3×30 mL), dichloromethane (3×30 mL). The resin wasthen suspended in N-methylpyrrolidinone (10 mL) andN-Fmoc-3-cyclohexyl-L-alanine (2.7 g, 6.93 mmol),diisopropylcarbodiimide (1.09 mL, 6.93 mmol) and HOBT (0.936 g, 6.93mmol) were added. The resulting mixture was shaken at ambienttemperature overnight and filtered. The resin was washed withN,N-dimethylformamide (3×100 mL), methanol (3×100 mL), dichloromethane(3×100 mL) and dried in vacuo.

[0097] Step (iii). To the resin product of the previous step (200 mg,0.14 mmol) was added 20% piperidine in N,N-dimethylformamide (5 mL) andthe reaction mixture was shaken at ambient temperature for 30 min. Themixture was filtered and the resin was washed with N,N-dimethylformamide(3×10 mL), methanol (3×10 mL), dichloromethane (3×10 mL). The resin wasthen suspended in N-methylpyrrolidinone (2 mL)N-Fmoc-2-amino-2-methylpropanoic acid (136 mg, 0.42 mmol),diisopropylcarbodiimide (65 μL, 0.42 mmol) and HOBT (57 mg, 0.42 mmol)were added. The resulting mixture was shaken at ambient temperatureovernight and filtered. The resin was washed with N,N-dimethylformamide(3×10 mL), methanol (3×10 mL), dichloromethane (3×10 mL) and dried invacuo.

[0098] Step (iv). To the product of the previous step (0.14 mmol) wasadded 20% piperidine in N,N-dimethylformamide (5 mL) and the reactionmixture was shaken at ambient temperature for 30 min. The mixture wasfiltered and the resin was washed with N,N-dimethylformamide (3×10 mL),methanol (3×10 mL), dichloromethane (3×10 mL). The resin was thensuspended in dichloromethane (2 mL) and treated withdiisopropylethylamine (73 μL, 0.42 mmol). The reaction mixture was thencooled to 0° C. and diphosgene (50 μL, 0.42 mmol) was added dropwise.The resulting mixture was allowed to warm to the ambient temperature andwas stirred for 3 h. The mixture was filtered and the resin was washedwith dichloromethane (3×10 mL) and dried in vacuo. The resin was thensuspended in toluene (2 mL) and stirred reaction mixture was heated at70° C. for 4 h. The cooled resin mixture was filtered and the resin waswashed with dichloromethane (3×10 mL). Cleavage from the support waseffected by treatment with 50% trifluoroacetic acid in dichloromethane(3 mL) for 1 hr. Concentration of the filtrate yielded a brown solid.

[0099] Step (v). Without further purification, the solid from Step (iv)was dissolved in methanol (1 mL) and then treated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (40 mg, 0.21 mmol). Themixture was stirred at ambient temperature overnight and then wasconcentrated in vacuo. The resulting oil was triturated with 99/1dichloromethane/methanol (3×5 mL) and filtered through a silica gelplug. The filtrate was concentrated in vacuo to afford(S)-2-[[3-cyclohexyl-2-(4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester (18 mg) as a white foam: EI-HRMS m/e calcd forC₁₉H₂₆N₄O₅S (M⁺) 423.1702, found 423.1701.

EXAMPLE 4

[0100] Preparation of(S,S)-2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0101] The compound was prepared as described in Example 3, exceptN-Fmoc-3-cyclohexyl-L-alanine was the amino acid incorporated in Step(iii) of the procedure. The title compound was obtained as a white foam:EI-HRMS m/e calcd for C₂₄H₃₄N₄O₅S (M⁺) 491.2328, found 491.2323.

EXAMPLE 5

[0102] Preparation of(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(naphthalen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0103] The compound was prepared as described in Example 3, exceptN-Fmoc-3-(naphthalen-2-yl)-L-alanine was the amino acid incorporated inStep (iii) of the procedure. The title compound was obtained as a whitefoam: EI-HRMS m/e calcd for C₂₈H₃₀N₄O₅S (M⁺) 535.2015, found 535.2035.

EXAMPLE 6

[0104] Preparation of2-[[(S)-2-[(R)-4-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-yl]-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0105] The compound was prepared as described in Example 3, exceptN-Fmoc-3-(4-chlorophenyl)-D-alanine was the amino acid incorporated inStep (iii) of the procedure. The title compound was obtained as a whitefoam: EI-HRMS m/e calcd for C₂₄H₂₇N₄O₅SCl (M⁺) 519.1469, found 519.1466.

EXAMPLE 7

[0106] Preparation of(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(4-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0107] The compound was prepared as described in Example 3, exceptN-Fmoc-L-tyrosine was the amino acid incorporated in Step (iii) of theprocedure. The title compound was obtained as a white foam: EI-HRMS m/ecalcd for C₂₄H₂₈N₄O₆S (M⁺) 501.1808, found 501.1815.

EXAMPLE 8

[0108] Preparation of(S)-2-[[3-cyclohexyl-2-(2,4-dioxo-1,3-diazaspiro[4.4]non-3-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0109] The compound was prepared as described in Example 3, exceptN-Fmoc-1-aminocyclopentanecarboxylic acid was the amino acidincorporated in Step (iii) of the procedure: EI-HRMS m/e calcd forC₂₁H₂₈N₄O₅S (M⁺) 449.1859, found 449.1853.

EXAMPLE 9

[0110] Preparation of(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(3-hydroxybenzyl)imidazolidin1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.

[0111] The compound was prepared as described in Example 3, exceptN-Fmoc-3-(3-hydroxyphenyl)-L-alanine was the amino acid incorporated inStep (iii) of the procedure: EI-HRMS m/e calcd for C₂₄H₂₈N₄O₆S (M⁺)501.1808, found 501.1816.

EXAMPLE 10

[0112] Preparation of2-[[(S)-3cyclohexyl-2-[(R,S)-2,5-dioxo-4-(4-fluorobenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0113] The compound was prepared as described in Example 3, exceptN-Fmoc-3-(4-fluorophenyl)-DL-alanine was the amino acid incorporated inStep (iii) of the procedure: EI-HRMS m/e calcd for C₂₄H₂₇N₄O₅SF (M⁺)503.1764, found 503.1776.

EXAMPLE 11

[0114] Preparation of(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(thiophen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0115] The compound was prepared as described in Example 3, exceptN-Fmoc-3-(thiophen-2-yl)-L-alanine was the amino acid incorporated inStep (iii) of the procedure: EI-HRMS m/e calcd for C₂₂H₂₆N₄O₅S₂ (M⁺)491.1423, found 491.1425.

EXAMPLE 12

[0116] Preparation of2-[[(S)-3-cyclohexyl-2-[(R)-2,5-dioxo-4-propylimidazolidin1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester

[0117] The compound was prepared as described in Example 3, except(R)-N-Fmoc-2-aminopentanoic acid was the amino acid incorporated in Step(iii) of the procedure: El-HRMS m/e calcd for C₂₀H₂₈N₄O₅S (M⁺) 437.1859,found 437.1850.

EXAMPLE 13

[0118] Preparation of(S,S)-2-[[2-(4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0119] The compound was prepared as described in Example 3, exceptN-Fmoc-L-phenylalanine was the amino acid incorporated in Step (iii) ofthe procedure: EI-HRMS m/e calcd for C₂₄H₂₈N₄O₅S (M⁺) 485.1859, found485.1857.

EXAMPLE 14

[0120] Preparation of(S,S)-2-[[3-cyclohexyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0121] The compound was prepared as described in Example 3, exceptN-Fmoc-3-cyclopentyl-L-alanine was the amino acid incorporated in Step(iii) of the procedure: El-HRMS m/e calcd for C₂₃H₃₂N₄O₅S (M⁺) 477.2172,found 477.2170.

EXAMPLE 15

[0122] Preparation of(S,S)-2-[[3-cyclopentyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.

[0123] The compound was prepared as described in Example 3, exceptN-Fmoc-3-cyclopentyl-L-alanine was the amino acid incorporated in bothStep (ii) and Step (iii) of the procedure. The title compound wasobtained as a white foam: EI-HRMS m/e calcd for C₂₂H₃₀N₄O₅S (M⁺)463.2015, found 463.2023.

EXAMPLE 16

[0124] Preparation of(S,S)-2-[[2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin- 1-yl]-3-cyclopentylpropanoyl]amino]thiazole-4-carboxylic acid methylester.

[0125] The compound was prepared as described in Example 3, exceptN-Fmoc-3-cyclopentyl-L-alanine and N-Fmoc-3-cyclohexyl-L-alanine werethe amino acids incorporated in Step (ii) and Step (iii) of theprocedure respectively: EI-HRMS m/e calcd for C₂₃H₃₂N₄O₅S (M⁺) 477.2172,found 477.2164.

EXAMPLE 17

[0126] Preparation of(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(thiazol-2-yl)propanamide:

[0127] Step (i). A mixture of N-Fmoc-3-cyclohexyl-L-alanine (3.47 g, 8.8mmol), 2,6-dichlorobenzoyl chloride (3.2 mL, 22 mmol) inN-methylpyrrolidinone (20 mL) was added into a fritted polypropylenecolumn charged with Wang resin (Midwest Bio-Tech, 4 g, 4.4 mmol). Thesuspension was shaken for 5 min, then pyridine (2.5 mL, 30.8 mmol) wasthen added slowly and the resulting dark mixture was shaken overnight atambient temperature. The mixture was then filtered and the resin waswashed with N,N-dimethylformamide (3×30 mL), methanol (3×30 mL),dichloromethane (3×30 mL) and dried in vacuo.

[0128] Step (ii). To the resin product of step (i) was added 20%piperidine in N,N-dimethylformamide (25 mL) and the reaction mixture wasshaken at ambient temperature for 30 min. The mixture was filtered andthe resin was washed with N,N-dimethylformamide (3×30 mL), methanol(3×30 mL), dichloromethane (3×30 mL). The resin was then suspended inN-methylpyrrolidinone (10 mL) and N-Fmoc-3-cyclohexyl-L-alanine (5.2 g,13.2 mmol), diisopropylcarbodiimide (2.1 mL, 13.2 mmol) and HOBT (1.8 g,13.2 mmol) were added. The resulting mixture was shaken at ambienttemperature overnight and filtered. The resin was washed withN,N-dimethylformamide (3×30 mL), methanol (3×30 mL), dichloromethane(3×30 mL) and dried in vacuo.

[0129] Step (iii). To the resin product of Step (ii) was added 20%piperidine in N,N-dimethylformamide (25 mL) and the reaction mixture wasshaken at ambient temperature for 30 min. The mixture was filtered andthe resin was washed with N,N-dimethylformamide (3×30 mL), methanol(3×30 mL), dichloromethane (3×30 mL). The resin was then suspended indichloromethane (20 mL) and treated with diisopropylethylamine (2.3 mL,13.2 mmol). The reaction mixture was then cooled to 0° C. and diphosgene(1.6 mL, 13.2 mmol) was added dropwise. The mixture was allowed to warmto room temperature with stirring for 5 h, then was filtered and theresin was washed with dichloromethane (3×30 mL) and dried in vacuo. Theresin was then suspended in toluene (20 mL) and the stirred mixture washeated at 70° C. for 4 h. The cooled resin mixture was filtered and theresin was washed with dichloromethane (3×30 mL). Cleavage from thesupport was effected by treatment with 50% trifluoroacetic acid indichloromethane (30 mL) for 1 hr. Concentration of the filtrate yieldeda brown solid. It was then purified by reversed phase HPLC to afford(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoicacid (850 mg) as a white foam: EI-HRMS m/e calcd for C₁₉H₃₀N₂O₄ (M⁺)350.2205, found 350.2204.

[0130] Step (iv). A solution of(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoicacid [Step (iii); 25 mg, 0.071 mmol] in N-methylpyrrolidinone (1 mL) wastreated with diisopropylethylamine (19 μL, 1.065 mmol) and HBTU (29.3mg, 0.078 mmol). The reaction mixture was then treated with2-aminothiazole (7.2 mg, 0.071 mmol) and stirred at ambient temperatureovernight. The reaction mixture was then diluted with water (2 mL) andextracted with ethyl acetate (2×3 mL). The combined organic layers weredried over magnesium sulfate, filtered, and concentrated in vacuo. Theproduct was purified by using flash chromatography (Merck Silica gel 60,230-400 mesh, 99/1 dichloromethane/methanol) to furnish(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(thiazol-2-yl)propanamide(27 mg, 88%) as a white foam: EI-HRMS m/e calcd. for C₂₂H₃₂N₄O₃S (M⁺)433.2273, found 433.2270.

EXAMPLE 18

[0131] Preparation of(S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazol-4-yl]oxoaceticacid ethyl ester.

[0132] By using the conditions described in Step (iv) of Example 17,ethyl 2-amino-4-thiazoleglyoxylate was condensed with(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidinyl]propanoicacid [Example 17, Step (iii)] to give the title compound as a colorlessfoam: EI-HRMS m/e calcd. for C₂₆H₃₆N₄O₆S (M⁺) 533.2434, found 533.2431.

EXAMPLE 19

[0133] Preparation of(S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazol-4-yl]aceticacid ethyl ester.

[0134] By using the conditions described in Step (iv) of Example 17,ethyl 2-amino-4-thiazoleacetate was condensed with(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidinyl]propanoicacid [Example 17, Step (iii)] to give the title compound as a colorlessfoam: EI-HRMS m/e calcd. for C₂₆H₃₈N₄O₅S (M⁺) 519.2641, found 519.2620.

EXAMPLE 20

[0135] Preparation of(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(5-methylpyridin-2-yl)propanamide.

[0136] By using the conditions described in Step (iv) of Example 17,2-amino-5-methylpyridine was condensed with(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidinyl]propanoicacid [Example 17, Step (iii)] to give the title compound as a colorlessfoam: EI-HRMS m/e calcd. for C₂₅H₃₆N₄O₃ (M⁺) 441.2866, found 441.2869.

EXAMPLE 21

[0137] Preparation of(S,S)-6-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]nicotinicacid methyl ester.

[0138] By using the conditions described in Step (iv) of Example 17,methyl 6-aminonicotinate was condensed with(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidinyl]propanoicacid [Example 17, Step (iii)] to give the title compound as a colorlessfoam: EI-HRMS m/e calcd for C₂₆H₃₆N₄O₅ (M⁺) 485.2764, found 485.2768.

EXAMPLE 22

[0139] Preparation of(S,S)-N-(5-chloropyridin-2-yl)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanamide.

[0140] By using the conditions described in Step (iv) of Example 17,2-amino-5-chloropyridine was condensed with(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidinyl]propanoicacid [Example 17, Step (iii)] to give the title compound as a colorlessfoam: EI-HRMS m/e calcd for C₂₄H₃₃N₄O₃Cl (M⁺) 461.2319, found 461.2321.

EXAMPLE 23

[0141] Preparation of(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(pyridin-2-yl)propanamide.

[0142] By using the conditions described in Step (iv) of Example 17,2-aminopyridine was condensed with(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidinyl]propanoicacid [Example 17, Step (iii)] to give the title compound as a colorlessfoam: EI-HRMS m/e calcd for C₂₄H₃₄N₄O₃ (M⁺) 427.2709, found 427.2706.

BIOLOGICAL ACTIVITY

[0143] Example A

[0144] Glucokinase Activity

[0145] Glucokinase Assay: Glucokinase (GK) was assayed by coupling theproduction of glucose-6-phosphate to the generation of NADH withglucose-6-phosphate dehydrogenase (G6PDH, 0.75-1 k units/mg; BoehringerMannheim, Indianapolis, Ind.) from Leuconostoc mesenteroides as thecoupling enzyme (Scheme 2).

[0146] Recombinant human liver GK1 was expressed in E. coli as aglutathione S-transferase fusion protein (GST-GK) [Liang, Y., Kesavan,P., Wang, L., Niswender, K., Tanizawa, Y., Permut, M. A., Magnuson, M.,and Matschinsky, F. M. Variable effects ofmaturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutationson the substrate interactions and stability of the enzyme. Biochem. J.309:167-173, 1995] and was purified by chromatography over aglutathione-Sepharose 4B affinity column using the procedure provided bythe manufacturer (Amersham Pharmacia Biotech, Piscataway, N.J.).Previous studies have demonstrated that the enzymatic properties ofnative GK and GST-GK are essentially identical (Liang et al, 1995; Neet,K., Keenan, R. P., and Tippett, P.S. Observation of a kinetic slowtransition in monomeric glucokinase. Biochemistry 29;770-777, 1990).

[0147] The assay was conducted at 25° C. in a flat bottom 96-well tissueculture plate from Costar (Cambridge, Mass.) with a final incubationvolume of 120 μl. The incubation mixture contained: 25 mM Hepes buffer(pH, 7.1), 25 mM KCl, 5 mM D-glucose, 1 mM ATP, 1.8 mM NAD, 2 mM MgCl₂,1 μM sorbitol-6-phosphate, 1 mM dithiothreitol, test drug or 10% DMSO,1.8 unit/mL G6PDH, and GK (see below). All organic reagents were >98%pure and were from Boehringer Mannheim with the exceptions of D-glucoseand Hepes that were from Sigma Chemical Co, St. Louis, Mo. Testcompounds were dissolved in DMSO and were added to the incubationmixture minus GST-GK in a volume of 12 μl to yield a final DMSOconcentration of 10%. This mix was preincubated in the temperaturecontrolled chamber of a SPECTRAmax 250 microplate spectrophotometer(Molecular Devices Corporation, Sunnyvale, Calif.) for 10 minutes toallow temperature equilibrium and then the reaction was started by theaddition of 20 μl GST-GK.

[0148] After addition of enzyme, the increase in optical density (OD) at340 nm was monitored over a 10 minute incubation period as a measure ofGK activity. Sufficient GST-GK was added to produce an increase in OD₃₄₀of 0.08 to 0.1 units over the 10 minute incubation period in wellscontaining 10% DMSO, but no test compound. Preliminary experimentsestablished that the GK reaction was linear over this period of timeeven in the presence of activators that produced a 5-fold increase in GKactivity. The GK activity in control wells was compared with theactivity in wells containing test GK activators, and the concentrationof activator that produced a 50% increase in the activity of GK, i.e.,the SC_(1.5), was calculated.

[0149] All of the compounds of Formula I described in the SynthesisExamples had an SC_(1.5) less than or equal to 30 μM.

1. A compound of the formula:

wherein R₁ is a five- or six- membered aromatic heterocyclic ring havingone to three heteroatoms selected from nitrogen, oxygen, and sulfur,which ring is unsubstituted or substituted with halo, amino,hydroxylamino, nitro, cyano, sulfonamido, lower alkyl, perfluoro loweralkyl, lower alkyl thio, perfluoro-lower alkyl thio, lower alkylsulfonyl, perfluoro-lower alkyl sulfonyl, lower alkyl sulfinyl, or—(R₅)_(n)-C(O)—OR₆; R₂ is a cycloalkyl ring containing from 5 to 7carbon atoms; R₃ is hydrogen, lower alkyl, a cycloalkyl ring containingfrom 5 to 7 carbon atoms, unsubstituted aryl, aryl substituted with haloor hydroxy, or an unsubstituted five- or six- membered aromaticheterocyclic ring having one or two heteroatoms selected from nitrogen,oxygen, and sulfur; R₄ is hydrogen, lower alkyl, or R₃ and R₄ togetherwith the carbon atom to which they are attached form a cycloalkyl ringcontaining 5 to 7 carbon atoms; R₅ is —C(O)— or lower alkyl; R₆ is loweralkyl; n is 0 or 1; * and * * each designate an asymmetric centers andpharmaceutically acceptable salts thereof.
 2. The compound of claim 1wherein R₁ is substituted or unsubstituted thiazolyl.
 3. The compound ofclaim 2 wherein R₁ is substituted thiazolyl.
 4. The compound of claim 3wherein R₁ is thiazolyl substituted with —(R₅)_(n)-C(O)—OR₆.
 5. Thecompound of claim 4 wherein R₂ is cyclopentyl or cyclohexyl.
 6. Thecompound of claim 5 wherein R₃ is cyclopentyl or cyclohexyl and R₄ ishydrogen.
 7. The compound of claim 6 wherein n is
 0. 8. A compound ofclaim 7 which is(S,S)-2-[[2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl)-3-cyclopentylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 9. A compound of claim 7 which is(S,S)-2-[[3-cyclopentyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 10. A compound of claim 7 which is(S,S)-2-[[3-cyclohexyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 11. A compound of claim 7 wherein R₂ and R₃ arecyclohexyl.
 12. A compound of claim 11 which is(S,S)-2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 13. A compound of claim 6 wherein R₂ and R₃arecyclohexyl.
 14. A compound of claim 13 which is(S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazol-4-yl]oxoaceticacid ethyl ester.
 15. A compound of claim 13 which is(S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazol-4-yl]aceticacid ethyl ester.
 16. A compound of claim 5 wherein R₃ is substituted orunsubstituted phenyl and R₄ is hydrogen.
 17. A compound of claim 16which is(S,S)-2-[[2-(4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 18. A compound of claim 16 which is2-[[(S)-2-[(R)-4-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-yl]-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylic acid methyl ester. 19.A compound of claim 16 which is(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(4-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 20. A compound of claim 16 which is(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(3-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 21. A compound of claim 16 which is2-[[(S)-3-cyclohexyl-2-[(R,S)-2,5-dioxo-4-(4-fluorobenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 22. A compound of claim 5 wherein at least one of R₃and R₄ are lower alkyl.
 23. A compound of claim 22 which is(S)-2-[[3-cyclohexyl-2-(4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 24. A compound of claim 22 which is2-[[(S)-3-cyclohexyl-2-[(R)-2,5-dioxo-4-propylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 25. A compound of claim 5 wherein R₃ is naphthyl andR₄ is hydrogen.
 26. A compound of claim 25 which is(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(naphthalen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 27. A compound of claim 5 wherein R₃ and R₄ togetherwith the carbon atoms to which they are attached form a cycloalkyl ringcontaining 5 to 7 carbon atoms.
 28. A compound of claim 27 which is(S)-2-[[3-cyclohexyl-2-(2,4-dioxo-1,3-diazaspiro[4.4]non-3-yl)propanoyl]amino]thiazole-4-carboxylicacid methyl ester.
 29. A compound of claim 5 wherein R₃ is anunsubstituted five- or six-membered aromatic heterocyclic ring havingone or two heteroatoms selected from nitrogen, oxygen, and sulfur.
 30. Acompound of claim 29 which is (S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(thiophen-2-yl)methylimidazolidin-1-yl]propanoyl]amino] thiazole-4-carboxylic acid methyl ester.
 31. A compound of claim2 wherein R₁ is unsubstituted thiazolyl.
 32. A compound of claim 31wherein R₂ and R₃ are cyclohexyl and R₄ is hydrogen.
 33. A compound ofclaim 32 which is (S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(thiazole-2-yl)propanamide.34. A compound of claim 1 wherein R₁ is substituted or unsubstitutedpyridine.
 35. A compound of claim 34 wherein R₂ is cyclohexyl.
 36. Acompound of claim 35 wherein R₃ is cyclohexyl and R₄ is hydrogen.
 37. Acompound of claim 36 wherein R₁ is substituted pyridine.
 38. A compoundof claim 37 wherein the pyridine is substituted with —(R₅)_(n)-C(O)—OR₆.39. A compound of claim 38 wherein n is 0 and R₆ is lower alkyl.
 40. Acompound of claim 39 which is(S,S)-6-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]nicotinicacid methyl ester.
 41. A compound of claim 37 which is(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(5-methylpyridin-2-yl)propanamide.42. A compound of claim 37 which is(S,S)-N-(5-chloropyridin-2-yl)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanamide.43. A compound of claim 36 wherein R₁ is unsubstituted pyridine.
 44. Acompound of claim 43 which is(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(pyridin-2-yl)propanamide.